ABSTRACT
Objective:To investigate the immunoregulatory effects of lentivirus-mediated microRNA (miR)-31-5p overexpression on peripheral blood T helper cell 17 (Th17) in a rabbit model of autoimmune dry eye.Methods:The miR-31-5p recombinant lentiviral vector was constructed.Lentivirus overexpressing miR-31-5p and its control virus were packaged.The concentration measurement and lentiviral titer determination were carried out.A rabbit model of autoimmune dry eye was established and the peripheral blood mononuclear cells (PBMC) of the rabbits were isolated.PBMC infected with miR-31-5p and negative control lentivirus particles were assigned as the miR-31-5p overexpression group and control group, respectively.The miR-31-5p expression level was detected using quantitative real-time PCR (qRT-PCR). Then PBMC in the two groups were co-cultured with γ-ray irradiated lacrimal gland epithelial cells.The expressions of Th17 cell related transcription factor retinoic acid-receptor-related orphan receptor C (RORC) and interleukin-17 (IL-17) mRNA, IL-1β, IL-6 and IL-23 were determined by qRT-PCR.The IL-17 protein expression level was detected by Western blot.The use and care of animals complied with Regulation for the Administration of Affair Concerning Experiment Animals by State Science and Technology Commission.The study protocol was approved by the Ethics Committee of Tianjin Medical University Eye Hospital (No.TJYY20201221036).Results:The construction of the miR-31-5p recombinant lentiviral vector was verified by DNA sequencing.The lentiviral titer of lentivirus overexpressing miR-31-5p and control lentivirus particles was 3.82×10 7 TU/ml and 3.50×10 7 TU/ml, respectively.The miR-31-5p relative expression level of PBMC was significantly increased in miR-31-5p overexpression group in comparison with control group, showing a statistically significant difference ( t=-9.696, P<0.001). When PBMC were co-cultured with lacrimal gland epithelial cells in vitro, the relative expression levels of RORC and IL-17 mRNA in miR-31-5p overexpression group were 0.33±0.03 and 0.28±0.09, which were significantly decreased in comparison with 1.00±0.00 and 1.00±0.00 in control group, with statistically significant differences between them ( t=46.256, 13.810; both at P<0.05). The relative expression level of IL-17 protein in miR-31-5p overexpression group was significantly reduced than control group ( t=4.977, P=0.008). The relative expression levels of IL-1β, IL-6 and IL-23 mRNA were significantly lower in miR-31-5p overexpression group than control group ( t=220.076, 6.641, 13.271; all at P<0.05). Conclusions:The overexpression of miR-31-5p can inhibit the Th17-immune response via down-regulating the expression of IL-6, IL-1β and IL-23.
ABSTRACT
To explore the role of miR-31-5p/STAT3 in the pathogenesis of colitis-assosiated cancer (CAC) and the intervention mechanism of Huangqi Baizhu decoction. Methods The CAC model of C57BL/6 mice was established by AOM/DSS method. The differential expression of MicroRNA in control and CAC mice was detected. qPCR was used to screen differential MicroRNAs; Western blot was used to detect the expression levels of STAT3 , p-STAT3 and IL-6Ra. Results AOM was injected into abdominal cavity once, combined with 3% DSS free drinking for three cycles to establish model. After eight weeks, moderate and/or severe dysplasia of colonic mucosa appeared in model mice. After detection, the expression of miR-31-5p and STAT3 and p-STAT3 proteins significantly increased. After intervention with Huangqi Baizhu decoction, the expressions of miR-31-5p and STAT3 and p-STAT3 proteins were down-regulated. In vitro, miR-31-5p over-expression in RAW264. 7 by transfect method, and the expression of STAT3 protein increased significantly compared with that of control. On the other hand, stimulated by IL-6 or TNF-a for 24 h, miR-31-5p levels were up-regulated significantly, suggesting that inflammatory factors could cause a positive correlation between miR-31-5p/STAT3. At the same time, ATRII + AST (atractylodesin II + astragaloside) mixture significantly down-regulated miR-31-5p, STAT3, IL-6Ra increase induced by IL-6 stimulation. Conclusions miR-31-5p/STAT3 forms a positive feedback loop, which plays an important role in the pathogenesis of CAC. miR-31-5p/STAT3 may continue to amplify the inflammation effect and eventually lead to the dysplasia of colonic epithelium and even tumorigenesis.
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@#Objective: To investigate the mechanism of miR-31-5p/tension protein 1 gene (TNS1) axis modulating radiotherapy resistance in breast cancer. Methods: The breast cancer tissues and corresponding para-cancerous tissues of 21 patients with breast cancer, who underwent surgical resection at Department of Cancer Radiotherapy of Nanyang Central Hospital from July 2017 to December 2017, were collected for this study; breast cancer cell lines (MCF-7，MDA-MB-23 and SKBR-3) were also collected; qPCR was applied to detect the expression level of miR-31-5p in breast cancer tissues and cell lines. The radiation resistant cell line MCF-7R was constructed by using 6 MV-X ray radiotherapy treatment. Subsequently, the influence of over-expression/kockdown of miR-31-5p on radiation sensitivity of MCF-7 and MCF-7R cells were detected by colony formation assay, Transwell assay and Annexin V-FITC/PI double staining flow cytometry assay, respectively. Moreover, luciferase reporter assay was used to verify whether TNS1 was a target gene of miR-31-5p. Results: Compared with para-cancerous tissues, normal mammary epithelial MCF-10A cells and MCF-7 cells, miR-31-5p was low-expressed in breast cancer, cell lines and MCF-7R (all P<0.01). Over-expression of miR-31-5p resulted in inhibited invasion and promoted apoptosis of MCF-7R cells (P<0.01), whereas miR-31-5p knockdown got opposite results in MCF-7 cells. Moreover, luciferase reporter assay confirmed that TNS1 was a target gene of miR-31-5p. Over-expression of miR-31-5p inhibited invasion and increased radio-sensitivity, apoptosis of MCF-7R cell via targeting TNS1 (P<0.01), whereas knockdown of miR-31-5p significantly promoted the invasion but reduced apoptosis of MCF-7R cells (all P<0.01), and further up-regulated the radio-sensitivity of MCF-7R cells. Conclusion: miR-31-5p/TNS1 axis regulates the radiotherapy resistance of breast cancer, and over-expression of miR-31-5p may reverse the resistance of MCF-7R to radiotherapy.
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Objective To study the expression and roles of miR-31-5p in acute myeloid leukemia (AML).Methods miR-31-5p in AML patients were evaluated by real-time PCR;THP-1 cells were transfected with the miR-31-5p mimic and control, respectively.The effects of over-expression of miR-31-5p were examined by CCK-8 and FACS analysis;Dual-luciferase and Western blot were performed to detect target gene HuR expression.The effects of knock-down of HUR were also examined by CCK-8 and FACS analysis.Results miR-31-5p was down-regulated in AML patients compared to the normal control.Over-expression of miR-31-5p in THP-1 cells reduces cell proliferation and accelerates monocytic differentiation.miR-31-5p could target HuR, and knock-down of HuR inhibits cell proliferation and attenuates monocytic differentiation in THP-1 cells.Conclusions miR-31-5p may regulate AML cell proliferation and monocytic differentiation by targeting HuR.